Speeding up for lympho-myeloid differentiation Free

In this issue of Blood, Wang et al report on a novel cell culture system that spotlights the close relationship between increasing proliferative activity and the concurrent lineage decision process in lympho-myeloid differentiation.¹ By integrating bulk and time-resolved single-cell colony assays with single-cell transcriptome analysis and mathematical modeling, the authors uncover coordinated patterns of accelerated proliferation during differentiation, and capture profiles of sustained cellular and transcriptional heterogeneity in human cells undergoing B-lymphoid (L) and neutrophil/monocyte (NM) lineage restriction.

The precise regulation of cell cycle progression and the onset of differentiation are essential for maintaining balanced blood cell production. Recent studies using L/NM-balanced in vitro systems suggest that human L and NM lineages originate from a common CD34⁺ progenitor with dual lineage restriction.² Although hematopoietic differentiation has traditionally been viewed as a stepwise process marked by a progressive loss of self-renewal and increased cell cycling,³ the exact timing and extent of these transitions during L and NM lineage restriction remain unclear. Increasing evidence suggests that lineage transitions occur through a continuum of heterogeneous cell states with dynamic gene expression changes,^4,5 yet it is still poorly understood how these changes are linked to intermediate stages of lineage commitment.

To explore these complexities, Wang and colleagues developed an advanced in vitro system with high cellular, temporal, and subcellular resolution and investigated how proliferative activity and molecular changes drive human L and NM lineage restriction. Starting from a population of human cord blood–derived P-mix cells (CD34⁺CD38^medCD71⁻CD10⁻) enriched in L+NM bilineage progenitors, they employed both feeder-free bulk assays and subsequent clonal assays and confirmed a strong link between specific surface markers and lineage commitment (CD45RA [RA] for L progenitors and CLEC12A [C] for NM progenitors), with double-negative cells retaining bilineage potential. Temporally resolved label dilution further confirmed that RA and C surface markers increase with cumulative and accelerated cell divisions, which is also validated by complementary cell cycle analyses.

To dissect the heterogeneity in proliferative and lineage potential in the P-mix population, the authors used a sophisticated live-cell imaging approach to track single-cell colonies over 16 days and study the temporal alignment of division dynamics and differentiation onset. Interestingly, they found that delays in accelerated proliferation correlated with delays in differentiation onset, suggesting that these processes are functionally linked. A complementary mathematical modeling approach supported the experimental findings and attributed the pronounced heterogeneity observed in clonal expansion to an intrinsic heterogeneity of the seeding population. Further dissecting the dynamical aspects of the differentiation process, they showed that slow-cycling cells are associated with a higher proportion of bilineage progenitors and fewer differentiated phenotypes compared with fast-cycling cells.

The authors complemented their dynamic assessments with single-cell transcriptome sequencing to identify characteristic changes during lineage restriction. Using pseudotime modeling they analyzed different lineage restriction trajectories and confirmed that self-renewal genes are shared in the most primitive cells, whereas differentiation-associated transcription factors were detected across multiple lineages. These results are in line with early findings from the 1990s that multipotent hematopoietic progenitors are “primed” for commitment through the coexpression of multiple lineage-associated genes.⁶ Combining these analyses with the assessment of cell cycle activity, the authors proposed a model in which proliferation activation precedes cell fate selection.

In summary, the study highlights the tight regulation between cell cycle activation and lineage restriction in bipotent hematopoietic progenitors. It shows that the actual lineage decision between L and NM fates is preceded by cell cycle shortening and decreasing expression of self-renewal associated genes. Cells with increased CD45RA expression commit to the lymphoid lineage (B cells), whereas those with higher CLEC12A expression follow the myeloid lineage (neutrophils and monocytes). This transition into progressive lineage restriction is orchestrated by dynamic shifts in signaling, epigenetics, and transcription.

From a methodological perspective, this work illustrates how time-resolved analyses in appropriate in vitro culture systems can dissect the dynamics of parallel processes during lineage commitment, thereby revealing the importance of a tight coordination between proliferation, self-renewal, and differentiation. Further investigations, both in vitro and in vivo, are needed to clarify how these processes are functionally linked and influenced by external factors such as hematopoietic microenvironmental cues.

Apart from the tightly regulated balance between cell cycle and differentiation, the authors found a pronounced level of heterogeneity within the P-mix cell population with respect to the cells’ lineage potential, their clonal expansion, and their transcriptional variability. Also, the supporting mathematical model is essentially built on the assumption of intrinsic cellular heterogeneity. These findings support Connie Eaves’s general perception that heterogeneity is a fundamental principle in hematopoiesis, essential for the functionality of blood formation and robustly re-established over time.^7,8 The question remains whether there is even a broader concept of stemness or multilineage potential, which is not the primary property of an individual cell but rather a systemic property of a heterogeneous cell population.⁹ From a conceptual perspective this notion better reflects different and potentially contrasting properties of stem cells (such as the defining dualism between self-renewal and differentiation) and their potential to respond to multiple environmental cues simultaneously. Revealing the origin of this robust heterogeneity in hematopoietic stem and progenitor populations is a quest for the coming decade.

It was Connie Eaves’s aim “to establish models that will accommodate such pluralistic features of HSCs and their control mechanisms.”⁸ The work by Wang et al is one of the last projects developed under her guidance and has succeeded in progressing along those lines. It stands as one of her legacies and is a remarkable conclusion to a life dedicated to advancements in hematology.

Conflict-of-interest disclosure: The authors declare no competing financial interests.